Our aim is to define the details of how the enhancers of the human virus, BKV, regulate the biology of the virus and to determine what role the BKV enhancers play in viral transformation and tumorigenesis. The enhancer element of BKV consists of three 68 bp tandem repeats which stimulate early viral promoter transcription. Recently, we have discovered that the BKV enhancers are necessary for viral replication. Also, reducing the number of BKV enhancers is known to increase the transformation efficiency of the virus. By extrapolation, it is likely that the BKV enhancers affect episomal persistence of the viral genome and tumorigenesis. Our approach will be to generate a variety of different enhancer mutations and assay for altered viral regulation, transformation and tumorigenesis. The types of enhancer element mutations that will be generated are: enhancer elements with one, and two repeats; 5' and 3' enhancer terminal deletions; internal deletions; site-specific mutations of interesting landmark sequences; reverse orientation; and direct substitution of BKV enhancers with those of other viruses. We will test the enhancer mutations both in the context of virus and plasmids, in which specific viral functions can be studied independently of one another. These viruses and plasmids will be tested in distinct assay systems to determine the effect of the mutated enhancers on viral replication, transcription, episomal persistence, transformation and tumorigenesis. We will also use a variety of techniques to explore the binding of specific proteins to the BKV enhancers with the aim of defining how these proteins may regulate the different enhancer functions. In particular, we will investigate the role of the TGGCA nuclear protein which specifically binds to the BKV enhancers. We will use our site- specific enhancer mutants to define the relationship between altered replication and binding of the TGGCA protein. We will also attempt to develop an in vitro replication system to further define the role of the BKV enhancers in replication.